This invention relates to the field of materials and methods for isolating biological entities, specifically, to materials and methods for isolating nucleic acids such as ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) from other material in a biological sample, such as cellular debris. More specifically, and without intending to limit the scope hereof, this invention particularly relates to the field of materials and methods for isolating RNA, more particularly to materials and methods for isolating total RNA from biological material such as animal or plant tissue, cultured tissue culture cells, yeast, bacteria, blood cells, viruses, or serum.
Many molecular biological techniques such as reverse transcription, cloning, restriction analysis, and sequencing involve the processing or analysis of biological materials. These techniques generally require that such materials be substantially free of contaminants capable of interfering with such processing or analysis procedures. Such contaminants generally include substances that block or inhibit chemical reactions, (e.g. nucleic acid or protein hybridizations, enzymatically catalyzed reactions, and other types of reactions, used in molecular biological techniques), substances that catalyze the degradation or de-polymerization of a nucleic acid or other biological material of interest, or substances that provide "background" indicative of the presence in a sample of a quantity of a biological target material of interest when the nucleic acid is not, in fact, present in the sample. Contaminants also include macromolecular substances from the in vivo or in vitro medium from which a nucleic acid material of interest is isolated, macromolecular substances such as enzymes, other types of proteins, polysaccharides, or polynucleotides, as well as lower molecular weight substances, such as lipids, low molecular weight enzyme inhibitors, or oligonucleotides. Contaminants can also be introduced into a target biological material from chemicals or other materials used to isolate the material from other substances. Common contaminants of this last type include trace metals, dyes, and organic solvents.
Obtaining nucleic acids, such as RNA or DNA, which are sufficiently free of contaminants for molecular biological applications is complicated by the complex systems in which the nucleic acids are typically found. These systems, e.g., cells from tissues, cells from body fluids such as blood, lymph, milk, urine, feces, semen, or the like, cells in culture, agarose or polyacrylamide gels, or solutions in which target nucleic acid amplification has been carried out, typically include significant quantities of contaminants from which the DNA or RNA of interest must be isolated before being used in a molecular biological technique.
Many different methods have been employed over the past several years to isolate target nucleic acids, such as DNA or RNA or specific types of DNA or RNA, from various different types of biological material. See, e.g. Chapter 2 (DNA) and Chapter 4 (RNA) of F. Ausubel et al., eds., Current Protocols in Molecular Biology, Wiley-Interscience, New York (1993). Conventional nucleic acid isolation protocols begin with the disruption of a sample of biological material under conditions designed to cause any target nucleic acid contained therein to be released into the disruption solution. Cells with a lipid bilayer membrane, such as bacteria cells, eukaryotic tissue culture cells, or blood cells are generally disrupted by being suspended in a solution and by adding a lysis buffer containing enzymes and/or chemicals designed to lyse the cells gently and to release the target nucleic acid into the solution. When RNA is the target nucleic acid to be isolated, biological material disruption is conventionally done under conditions designed to inhibit enzymes such as ribonucleases (RNases) capable of degrading RNA. One conventional way of inhibiting RNases during cell lysis and the initial processing steps is to include a guanidine salt, such as guanidine thiocyanate, and .beta.-mercaptoethanol in the disruption solution. Chirgwin (2979) Biochemistry 18:5294. Once the biological material is sufficiently disrupted to release the target nucleic acid material into the disruption solution, the resulting solution is generally spun in a centrifuge to remove at least some of the cell debris and any precipitates formed in the disruption solution during the disruption step. The supernatant is then decanted and processed further to separate the target nucleic acid material from other contaminants in the solution.
Conventional nucleic acid isolation protocols also generally use phenol or an organic solvent mixture containing phenol and chloroform to extract cellular material, such as proteins and lipids, remaining in the disruption solution supernatant produced as described above The phenol/chloroform extraction step is followed by precipitation of the nucleic acid material remaining in the extracted aqueous phase by adding alcohol, such as ethanol or isopropanol, to that aqueous phase. The precipitate is typically removed from the solution by centrifugation, and the resulting pellet of precipitate is allowed to dry before being resuspended in water or a buffer solution for further processing or analysis. The alcohol precipitation step serves two purposes in a conventional nucleic acid isolation procedure. Specifically, it allows one further to isolate the nucleic acid material from contaminants including residual phenol or chloroform remaining in the organic phase, and it allows one to resuspend the resulting precipitated nucleic acid in any solution for any final desired nucleic acid concentration. For examples of conventional lysis and organic extraction methods for isolating total RNA from various types of biological material, see: Molecular Cloning by Sambrook et al., 2nd edition, Cold Spring Harbor Laboratory Press, p. 7.3 et seq (1989); Protocols and Applications Guide produced by Promega Corporation, 3rd edition, p. 93 et seq. (1996); and by Chirgwin, J. M. et al, 18 Biochemistry 5294 (1979); all of which are incorporated by reference herein. Several different companies, including Promega Corporation (Madison, Wis., USA), have also developed kits which include reagents designed to be used in isolating RNA or mRNA from biological material using such methods of isolation. See, e.g. the RNAgents.RTM. Total RNA Isolation Systems and the PolyATract.RTM. Systems available from Promega and described in the 1996 Promega Product Catalogue at page 174.
Conventional nucleic acid isolation procedures have significant drawbacks. Among these drawbacks are the time required for the multiple extraction steps needed to isolate any given nucleic acid material from other materials present in a solution produced from disrupting a biological material. For example, multiple extraction steps are required to isolate RNA from proteins, lipids, and chromosomal DNA, all of which are present in a solution produced from disrupting tissue culture cells or plant or animal tissue. Another drawback of conventional nucleic and isolated procedures is the need to use phenol and chloroform. Phenol is a known carcinogen, which causes severe burns on contact with skin. Chloroform is highly volatile, toxic and flammable. Another undesirable characteristic of organic extractions with phenol is that the oxidation products of phenol can damage nucleic acids. Only freshly redistilled phenol can be used, and nucleic acids cannot be left in the presence of phenol. Finally, some of the nucleic acid material is inherently lost at each organic extraction step as well as in the alcohol precipitation stage. Consequently, even under the best circumstances such conventional methods are time consuming, hazardous, and produce relatively low yields of isolated nucleic acid material. Also, the resulting isolated nucleic acid material is frequently contaminated with impurities, particularly with organic solvents, alcohol, and/or non-target nucleic acid material (e.g. chromosomal DNA contaminant from an RNA isolation procedure).
When a target nucleic acid of a particular molecular weight or size is required for additional analysis, molecular biologists frequently use polyacrylamide or agarose gel electrophoresis to fractionate total RNA or DNA or mixtures of RNA and DNA isolated according to one of the conventional procedures described above. The DNA or RNA is sometimes processed prior to fractionation by electrophoresis using conventional methods to produce RNA or DNA of the desired size or which includes a particular sequence of interest, e.g. by using the polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), or linker attachment. For analysis or treatment by a molecular biological procedure after fractionation, the RNA or DNA of interest in the fraction(s) must be separated from contaminants, such as agarose, other polysaccharides, polyacrylamide, acrylamide, or acrylic acid, in the gel used in such electrophoresis. Conventional procedures similar to those used to isolate total RNA or DNA as described above have also been developed to isolate nucleic acid material from such contaminants in electrophoresis gels. For example, when the nucleic acid material of a desired molecular weight is fractionated on an agarose gel, the band on the gel containing the desired material can be cut out of the gel and processed as follows. The excised band is then processed by digesting the agarose in the bank using an Agarase enzyme, or processed by removing the nucleic acid material from the excised band of gel material by electroelution. Whether digestion or electroelution is used to separate the nucleic acid material of interest from the gel, the nucleic acid material is conventionally isolated further using multiple organic extraction steps using phenol and chloroform, as described above, followed by alcohol precipitation used to isolate the resulting nucleic acid material from the agarose or other contaminates in the gel. Such conventional fractionation, digestion, and isolation procedures are very time consuming and suffer from the same disadvantages of low yield and impurities from organic and alcohol contaminants as are common in the conventional general nucleic acid isolation methods described above.
Silica based nucleic acid isolation techniques have been developed as alternatives to, or in addition to, the conventional isolation techniques described above. In these alternative techniques, silica materials, such as glass particles, glass powder, silica particles, glass microfibers, diatomaceous earth, and mixtures of the above have been employed in combination with aqueous solutions of chaotropic salts to isolate DNA, particularly plasmid DNA. See U.S. Pat. No. 5,075,430 and references cited therein, including Marko et al., Anal. Biochem. 121, 382-387 (1982) and Vogelstein et al., Proc. Natl. Acad. Sci. (USA) 76, 615-619 (1979). See also Boom et al., J. Clin. Microbiol. 28, 495-503 (1990). With reference to intact glass fiber filters used in combination with aqueous solutions of a chaotropic salt to separate DNA from other substances, see Chen and Thomas, Anal. Biochem. 101, 339-341 (1980). Numerous commercial sources offer silica-based matrices designed for use in isolating DNA using either centrifugation or vacuum filtration. See, e.g. Wizard.TM. Plus SV DNA purification systems line of products from Promega Corporation; or the QiaPrep.TM. line of DNA isolation systems from Qiagen Corp. (Chatsworth, Calif., U.S.A.).
Silica-based systems and methods have also been developed in recent years for use in isolating total RNA from at least some types of biological materials. For example, one company has developed an RNA isolation kit wich uses a glass fiber filter in a spin filter basket and a hybrid lysis buffer/binding solution with a high concentration of guanidine hydrochloride, a choatropic agent, to isolate total RNA from simple biological materials, such as cultured cells, blood, yeast, and bacteria. See, e.g. Product Insert for the High Pure RNA Isolation Kit (Catalog Number 1828 665) from Boehringer-Mannheim GmbH (Mannheim, Germany). Another company has developed a system for isolating total RNA from bacteria cells and tissue using a spin basket with a silica gel-based membrane, and a lysis buffer/binding solution containing guanidinium isothiocyanate. See, e.g. the RNeasy.TM. Total RNA Kit from QIAGEN Inc. (Chatsworth, Calif., U.S.A.), as described in the December 1994 RNeasy.TM. Handbook from QIAGEN. Both commercial systems described briefly above allow one to isolate total RNA quickly, but the yield and purity of RNA isolated thereby tends to be low, particularly when used to isolate RNA from complex biological materials, such as plant or animal tissue.
Known silica-based RNA isolation techniques employ the same basic sequence of steps to isolate target RNA from any given biological material. However, the concentrations and amounts of the various solutions used in each such procedure vary depending upon the composition of the silica-based matrix used in the method. The basic sequence of steps used in all known silica-based RNA isolation process consists of disruption of the biological material in the presence of a lysis buffer, followed by formation of a complex of nucleic acid(s) and a silica matrix, followed by removal of the lysis buffer mixture from the resulting complex and washing of the complex, followed by elution of the target nucleic acid from the complex. The steps of the basic known silica-based RNA isolation techniques are reviewed in greater detail below, with particular emphasis on the materials and methods used to isolate total RNA from biological material using the two commercial silica-based RNA isolation kits cited above, according to the manufacturer's instructions.
A chaotropic salt is included in the lysis buffer used in the first, disruption, step of the silica-based RNA isolation techniques to protect the RNA formerly contained in the biological material from enzymatic degradation during and after disruption. The species and amount of chaotropic salt in the lysis buffer is chosen to promote binding of the RNA in the resulting lysate to whatever form of silica matrix is used in the next step of the technique. For example, a lysis/binding buffer containing guanidine hydrochloride, a chaotropic salt, and a Triton.RTM. X-100 (a non-ionic detergent) is provided for use in disrupting biological material and promoting binding to the silica matrix in the spin columns sold with the High Pure RNA Isolation Kit (Boehringer Mannheim GmbH). The lysis/binding buffer provided with the RNeasy.TM. Total RNA Kit (QIAGEN Inc.) for use in the same basic disruption step contains guanidinium isothiocyanate, a similar chaotropic salt.
Disruption of a sample of biological material is followed immediately by centrifugation to remove particulate cell debris from the lysate. The High Pure RNA Isolation Kit is a exception to this general rule, and calls for proceeding immediately from lysis to application of the crude lysate to the spin column provided with that kit. For methods of isolation, including the method used with the RNeasy.TM. Total RNA Kit, which do include the preprocessing step of centrifugation of a crude lysate prior to contact with a silica matrix, the amount of cell debris typically removed in the preprocessing step is generally very low, particularly in comparison to the amount of impurities inherently present in a lysate solution. Many of the impurities remaining in the solution, particularly protein, lipid, and chromosomal DNA can clog the silica matrix and compete with binding of RNA species to the matrix in the next step of the method. Some of those impurities, particularly proteins and chromosomal DNA can also co-elute from the matrix with RNA in the final elution step of the method, below. Lysis of complex biological materials, such as animal or plant tissue, releases a large number of contaminants in to the lysis solution in the first step of the general silica based RNA isolation procedure described above. The higher the concentration of contaminants in the lysate solution, the more likely the contaminants will clog the silica matrix. Some silica based matrices are so sensitive to clogging that they cannot be used to process complex biological material, regardless of whether one pre-clarifies the lysate solution by centrifugation. See, e.g. High Pure RNA Isolation Kit from Boehringer Mannheim, which only comes with instructions for use in isolating RNA from cultured cells, blood, yeast, and bacteria, and which tends to clog to the point of being unusable when used to isolate RNA from more complex tissue.
The silica matrix used to isolate RNA in such procedures is generally in the form of silica material impregnating or coating a filter, in the form of a resin, or in the form of magnetic beads coated with silica. Regardless of the form in which it occurs, the silica matrix in the general procedure described above is placed in a filter basket, before or after being bound to nucleic acid materials in the lysate solution. The filter basket is typically shaped like a hollow tube, with an open inlet end and a closed base end. The filter basket contains at least one filter fitted into the inside of the basket at its base end, and the base has openings which allow solutions passing through the filter to flow out of the basket through the openings. The filter fitted into the base of the basket is designed to retain the silica material in the basket, and to permit solutions of disrupted biological material to pass through the filter into the collection tube when the basket is subjected to an external force, such as centrifugation or a vacuum.
Commercial filter baskets are typically designed to fit into a standard sized microcentrifuge collection tube in such a way that the basket/tube assembly fit inside a standard microcentrifuge. Some filter baskets are also designed to fit into a collection tube as well as being designed to form a substantially air tight seal with a vacuum manifold, either directly through an adapter which is a part of the base of the filter basket, or indirectly by fitting into a separate vacuum manifold adapter. For an example of this last type of filter basket and separate manifold adapter assembly, see the Wizard.RTM. Plus SV Minipreps Spin Columns and manifold adapters from Promega Corp. For an example of filter basket assemblies designed to be used only with spin filtration, see the spin columns provided with the two commercial RNA isolation kits described above, i.e. the RNeasy.TM. Total RNA Kit (QIAGEN, Inc.) and the High Pure RNA Isolation Kit (Boehringer Mannheim GmbH).
In the next stage of known silica-based RNA isolation methods, the lysate solution is placed into contact with a silica matrix in the presence of a sufficiently high concentration of a chaotropic salt to promote the binding of RNA with the silica matrix. Known silica-based RNA isolation procedures, including both commercial procedures described above, include a sufficient amount of chaotropic salt to promote binding to the silica matrix in the lysate solution created in the first step of the procedure. Once placed in contact with the silica matrix, the lysate solution is removed therefrom, using external force, such as centrifugation or vacuum filtration. As noted above, this solution removal process can be difficult when the concentration of contaminants in the lysate solution placed in contact with the matrix is high, such as are generally present in lysates of complex biological materials.
Once the lysate solution is removed from the silica matrix, the matrix is washed in a series of wash steps, involving applying a wash solution to the matrix and removing it therefrom.
The High Pure RNA Isolation Kit (Boehringer Mannheim GmbH) includes reagents designed to treat the silica matrix after removal of the lysate solution as described above, before beginning the wash steps of that procedure. Specifically, the High Pure RNA Isolation Kit includes lyophilized DNase I and a DNase incubation buffer consisting of 1 M NaCl, 20 mM Tris-HCl and 10 mM MnCl.sub.2, pH 7.0 (25.degree. C.). The kit instructions call for suspending the lyophilized DNase in water, and adding the suspended DNase and incubation buffer directly to the silica matrix, the glass fiber filter in the base of the spin filter basket provided with that kit, and allowing the solution to incubate in contact with the filter for 15 minutes. A first wash buffer containing guanidine hydrochloride and ethanol is then added to the filter basket, and removed by centrifugation. That first wash step is then followed by a second and third wash step with a second wash solution which contains only a buffer, salt (not a chaotropic salt) and ethanol.
The DNase treatment step in the High Pure RNA Isolation Kit procedure uses a high concentration sodium chloride salt solution. Unfortunately, DNase I is considerably less active in such high salt solutions, and therefore more enzyme must be added to digest the same amount of DNA compared to what would be required in a lower salt buffer. By adding larger amounts of DNase to the silica matrix, one increases the number of contaminants which must be removed from the matrix before the final elution step.
Once the wash steps in a silica based isolation procedure are complete, the RNA is eluted from the silica matrix using an elution buffer, usually a low salt buffer or water. The buffer is applied to the matrix, and then removed from the matrix into a sterile collection tube by centrifugation. Unfortunately, protein and other contaminants tend to co-elute from the two commercial silica matrices when used to isolate RNA according to the manufacturers' instructions provided with the two commercial RNA isolation kits described above. Also, the yield of RNA isolated using High Pure Isolation Kit spin columns tends to be low compared to yields obtained using other techniques and systems. RNA eluted from the RNeasy.TM. Total RNA Kit spin columns tends to be contaminated with chromosomal DNA. See Example 6 and FIG. 3, below, for test results illustrating the chromosomal contamination problem with this particular kit and associated method of isolation.
What is needed is a method or methods for isolating RNA from a sample of biological material, whereby the RNA isolated thereby is substantially free of contaminants, including proteins, lipids, genomic DNA, and any chemicals likely to inhibit or interfere with processing or analysis of the isolated RNA. The present invention provides methods for isolating RNA which is substantially free of such contaminants, using the types of silica matrices and filter basket assemblies and general isolation techniques described above. The RNA isolation method of the present invention produces a high yield of RNA, and is less labor intensive than conventional RNA isolation techniques. The RNA isolated in a practice of the method of the present invention is particularly well suited to such contamination sensitive subsequent molecular biology applications as cDNA library construction, reverse transcriptase polymerase chain reaction (RT-PCR), and as a substrate in various other forms of analysis. Many other applications of RNA separated or isolated using the present invention will be apparent to one skilled in the art.